Objective: This study aimed to evaluate the direct anti-dyskinesia effect of deep brain stimulation (DBS) of subthalamic nucleus (STN) on levodopa-induced on-dyskinesia in Parkinson's disease (PD) patients during the early period after surgery without reducing the levodopa dosage.Methods: We retrospectively reviewed PD patients who underwent STN-DBS from January 2017 to October 2019 and enrolled patients with levodopa-induced on-dyskinesia before surgery and without a history of thalamotomy or pallidotomy. The Unified Dyskinesia Rating Scale (UDysRS) parts I+III+IV and the Unified Parkinson's Disease Rating Scale part III (UPDRS-III) were monitored prior to surgery, and at the 3-month follow-up, the location of active contacts was calculated by postoperative CT–MRI image fusion to identify stimulation sites with good anti-dyskinesia effect.Results: There were 41 patients enrolled. The postoperative levodopa equivalent daily dose (LEDD) (823.1 ± 201.5 mg/day) was not significantly changed from baseline , while the UDysRS on-dyskinesia subscores significantly decreased from 24 (10–58) to 0 (0–18) [median (range)] after STN stimulation (P < 0.0001). The levodopa-induced on-dyskinesia recurred in stimulation-off/medication-on state in all the 41 patients and disappeared in 39 patients when DBS stimulation was switched on at 3 months of follow-up. The active contacts which correspond to good effect for dyskinesia were located above the STN, and the mean coordinate was 13.05 ± 1.24 mm lateral, −0.13 ± 1.16 mm posterior, and 0.72 ± 0.78 mm superior to the midcommissural point.Conclusions: High-frequency electrical stimulation of the area above the STN can directly suppress levodopa-induced on-dyskinesia. Dyskinesia is one of the most troublesome symptoms of advanced Parkinson's disease (PD), often induced by long-term dopaminergic treatment . Following the definition in the Unified Dyskinesia Rating Scale (UDysRS) , LID is Deep brain stimulation (DBS) of subthalamic nucleus (STN) could reduce the required levodopa dosage for symptom control , 4, and The DBS strategies could be different for on-dyskinesia and off-dystonia: stimulating the sensorimotor region could significantly improve cardinal parkinsonian symptoms   and alsoWe retrospectively reviewed the clinical records of 146 PD patients who underwent STN-DBS by the same two neurosurgeons (Zhang and Li) at the Xuanwu Hospital of Capital Medical University from January 2017 to October 2019. Patients who suffered from preoperative levodopa-induced on-dyskinesia and with a score of Unified Parkinson's Disease Rating Scale (UPDRS)  ≥1 were included, and patients who had a history of thalamotomy or pallidotomy, which may suppress LID, were excluded. Eventually, 41 patients were included in this study. Of the 41 patients, 23 were female and 18 were male. Their mean age was 62.7 ± 8.2 years. The mean duration of disease before the surgery was 10.4 ± 3.7 years. Forty patients presented with peak-dose dyskinesia and 1 patient (P2) with square-wave dyskinesia. Thirty-three patients had bilateral on-dyskinesia and 8 patients had unilateral dyskinesia at baseline . All theDBS electrode implantation was performed under local anesthesia. The CRW stereotactic frame  was applied under local anesthesia, then CT scanning was performed. The CT images were fused immediately with the preoperative magnetic resonance imaging  images through the StealthStation Surgical Navigation System , and the coordinates of the target and the entrance trajectory were defined on stereotactic MRI images by directly visualizing the STN. Intraoperative microelectrode single needle recording (MER) using the Microdrive system  was performed, starting from 10 mm above the target. After the precise localization of the target point, DBS electrodes  with four contacts were placed in such a way that the metal tip of the DBS electrode was located 2–3 mm above the ventral STN border, and the contacts were positioned and labeled as follows: contacts 0 and 1, inside the STN; contact 2, dorsal margin of the STN; and contact 3, above the STN. Then, the DBS electrodes were tunneled and connected to a rechargeable implantable pulse generator  implanted in the subclavian region under general anesthesia. Postoperative CT images were fused with the preoperative MRI images to confirm the final position of the electrode metal tip and the trajectory of the DBS electrode and to calculate the coordinates of each contact, and the distance from the metal tip to the center of each contact  was 0.75, 2.75, 4.75, and 6.75 mm, respectively.DBS programming was initiated within 3 days after surgery with an initial setting of 60–90 μs/130–160 Hz/1.0–1.5 V. Patients underwent adjustment of stimulation settings until optimal control of the symptoms was established during hospitalization. The adjustment strategy of DBS programming was as follows: firstly, we used unipolar stimulation and chose the contact which positioned at the dorsal margin of the STN as the active contact for patients with LID and the contact inside the STN for patients without LID; if the patient did not achieve good control of symptoms, then it was changed to dual-contact monopolar stimulation (a contact within the STN + a contact above the STN); finally, interleaving stimulation was utilized, when necessary.Patients had the first postoperative clinical assessments and adjustment of stimulation settings and medication in the 3-month follow-up. On the 1st day of follow-up, the stimulation parameters were carefully screened following all contacts in medication-off (Med-off) state after at least 12 h without taking any anti-parkinsonian medication in the morning; the contacts and stimulation parameters were optimized to obtain maximum clinical benefit and minimal side effects. After switching off DBS for 30 min, patients took the usual first morning dose of levodopa; if on-dyskinesia occurred, then we switched on the DBS to test the anti-dyskinesia effect of active contacts.* 100%). Student's t-test or the Wilcoxon signed-rank test was used to determine whether there was a significant difference between the clinical scale scores at baseline and at 3 months follow-up. Statistical analysis was performed with SPSS . P < 0.05 were considered statistically significant.The outcome assessments consisted of the on-dyskinesia subscores of the UDysRS (parts I+III+IV) and UPDRS-III before surgery and 3 months after surgery. Baseline assessments of UPDRS-III were completed in Med-off state after at least 12 h without taking any anti-parkinsonian medication, and UPDRS-III of the Med-on state was the maximum improvement following a dose of levodopa equal to 150% of the patient's usual first morning dose. At the 3-month follow-up, all scores were assessed in DBS stimulation-on (Stim-on) condition on the 2nd day following the same dose of levodopa as baseline. The clinical improvement was computed as ([(Prescores – Postscores)/Prescores] P = 0.348)  (823.1 ± 201.5 mg/day) was not significantly changed from the baseline  . There wP < 0.0001; However, the UDysRS on-dyskinesia subscores significantly reduced after STN-DBS stimulation [from baseline 24 (10–58) to 0 (0–18), median (range), P < 0.0001) and a 69.0 ± 12.4% improvement in Med-on/Stim-on relative to the Med-off at baseline   .Z-axis) of the electrode metal tip were −7.50, −7.71, −8.31, and −7.65 mm, respectively, inferior to the midcommissural point. P41 underwent dorsal relocation of bilateral DBS electrodes on the 6th day postoperatively by withdrawing the left DBS electrode 4 mm and the right electrode 2 mm ; the mean coordinate of these 72 contacts was 13.05 ± 1.24 mm lateral, −0.13 ± 1.16 mm posterior, and 0.72 ± 0.78 mm superior to the midcommissural point, and the majority (85%) were above the anteroposterior commissure plane , which were located above 1 mm inferior to the anteroposterior commissure plane (re plane . The unire plane .There were 55 contacts of 31 electrodes (19 patients) that were found to induce dyskinesia (SID) , which wX, Y, Z −12.35, −5.74, −8.31 mm) and right . Bilateral DBS electrodes were dorsally repositioned under a local anesthetic on the 6th day postoperatively by withdrawing the left DBS electrode 4 mm and the right electrode 2 mm, and there was sustained relief of dyskinesia using the most dorsal contacts without levodopa reduction (There was one patient (P41) who continued to experience persistent bilateral levodopa-induced on-dyskinesia after DBS stimulation by using the most dorsal contacts, even reducing the medication from Madopar 125 mg every 4 hours(q4h) to 62.5 mg q4h, and the coordinates of the electrode metal tip relative to the midcommissural point were left  who continued to experience persistent dyskinesia: SRD for two patients  and it finally self-resolved between 4 and 6 months postoperatively; SID for one patient (P27) and it was completely relieved after 1 year postoperatively when his tremor became less prominent and a good effect was obtained under the SID threshold; and the remaining two patients had unilateral levodopa-induced on-dyskinesia , which was finally completely relieved after levodopa reduction.In our study, from the overall level, we found that the LEDD after surgery was not significantly changed from baseline, but UDysRS on-dyskinesia subscores significantly decreased; from the individual level, we found that levodopa-induced on-dyskinesia recurred in Stim-off/Med-on state and disappeared when DBS stimulation was switched on in 39/41 (95%) patients at the 3-month follow-up. All these findings confirm that STN-DBS stimulation can directly suppress levodopa-induced on-dyskinesia.Z = −1), where the dorsal margin of the STN is estimated by microrecording (Z = 0), and the average vertical coordinate (Z-axis) was 0.72 mm superior to the midcommissural point (Z = +0.72), which is dorsally compared with the dorsal margin of the STN and consistent with Yoichi's observations , we carefully observed the relationship between dyskinesia, medication, and DBS stimulation to subdivide the type of postoperative dyskinesia. After STN-DBS, especially during the early postoperative period, dyskinesia could be complicated. Besides LID, two new types of dyskinesia came out: (1) SID –20, whicHigh-frequency electrical stimulation of the area above the STN can directly suppress levodopa-induced on-dyskinesia, and the STN-DBS strategy for PD patients with levodopa-induced on-dyskinesia is simultaneously stimulating both the sensorimotor region of the STN and the area above the STN. The depth of electrode insertion for STN-DBS to provide adequate coverage of the above STN is the crucial point for dyskinesia suppression.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the ethics committee of Xuanwu Hospital of Capital Medical University. The patients/participants provided their written informed consent to participate in this study.JL was the major contributor in writing the manuscript and contributed to the DBS programming. SM contributed to the diagnosis and clinical assessment of the patients. JL and YZ contributed to DBS surgery. XJ contributed to data acquisition. SM and YZ contributed to the manuscript editing. YZ was the guarantor of integrity of the entire study. All the authors had collectively poured in a lot of efforts into this study, read, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The nasal epithelium is a key portal for infection by respiratory viruses such as SARS-CoV-2 and represents an important target for prophylactic and therapeutic interventions. In the present study, we test the safety and efficacy of a newly developed nasal spray  against infection by SARS-CoV-2 and its Delta variant on an in vitro 3D-model of the primary human nasal airway epithelium. Safety was assessed in assays for tight junction integrity, cytotoxicity and cilia beating frequency. Efficacy against SARS-CoV-2 infection was evaluated in pre-viral load and post-viral load application on airway epithelium. No toxic effects of AM-301 on the nasal epithelium were found. Prophylactic treatment with AM-301 significantly reduced viral titer vs. controls over 4 days, reaching a maximum reduction of 99% in case of infection from the wild-type SARS-CoV-2 variant and more than 83% in case of the Delta variant. When AM-301 administration was started 24 h after infection, viral titer was reduced by about 12-folds and 3-folds on Day 4. The results suggest that AM-301 is safe and significantly decelerates SARS-CoV-2 replication in cell culture inhibition assays of prophylaxis  and mitigation . Its physical  mechanism of action, safety and efficacy warrant additional investigations both in vitro and in vivo for safety and efficacy against a broad spectrum of airborne viruses and allergens. The COVID-19 pandemic has had a massive toll on daily life ,3,4. TheSARS-CoV-2 infection is mainly contracted from airborne virions ,12. UponAn interesting approach to develop simple and safe interventions of this type is based on enhancing the protective function that the nasal barrier already exerts. Indeed, the airway epithelium of the nasal mucosa works as a physical barrier through the production of mucus, which traps pathogens. Thereafter, the clearing action of cilia discharges the mucus into the nasopharynx from where it is eventually swallowed . A secon®-based nasal spray, Nasaleze® Cold & Flu blocker, Vicks® First Defence Cold Virus Blocker), and recent research has investigated the possibility of repurposing these sprays as COVID-19 prophylactic agents. A previous study [Several nasal sprays are commercially available for the treatment of respiratory infections such as common cold and influenza  with the aim of providing a safe and effective means of protection against harmful airborne particles. Seeking to minimize potential side effects and to facilitate frequent and compliant use, AM-301 is composed of only inert ingredients such as pharmaceutical excipients and substances that are generally recognized as safe. Because the components are neither metabolized nor absorbed and the mechanism of action is physical, not chemical, AM-301 is a non-pharmaceutical medical device from a regulatory perspective. This study tests the ability of AM-301 to prevent SARS-CoV-2 infection or mitigate existing infections in vitro using a model of primary human nasal airway epithelium .2) for all inserts, indicating their suitability for use in toxicology testing. Mean values for inserts treated with AM-301 or the matrix and for untreated inserts increased slightly over 4 days. These results indicate that the product did not have any adverse effects on tissue integrity  = 29.30, p = 0.0008; time, F = 153.0, p < 0.0001; interaction F = 15.76, p < 0.0001. Post hoc Tukey’s test: Day −3: untreated vs. matrix, p > 0.05; untreated vs. AM-301, p > 0.05; matrix vs. AM-301, p > 0.05; Day 1: untreated vs. matrix, p > 0.05; untreated vs. AM-301, p = 0.0143; matrix vs. AM-301, p = 0.0264; Day 2: untreated vs. matrix, p = 0.0057, untreated vs. AM-301, p > 0.05, matrix vs. AM-301, p = 0.0005; Day 3: untreated vs. matrix, p > 0.05, untreated vs. AM-301, p = 0.0049, matrix vs. AM-301, p = 0.0210; Day 4, untreated vs. matrix, p = 0.0476; untreated vs. AM-301, p = 0.0082, matrix vs. AM-301, p > 0.05).Inserts of a 3D model of the primary human nasal airway epithelium (MucilAir) were repeatedly exposed to AM-301 or its matrix (lacking bentonite), or left untreated for 4 days, and then tested in four standard assays to evaluate MucilAir integrity. TEER measurements A at baset-test, matrix vs. AM-301: Day 2 (p = 0.776); Day 4 (p = 0.392). These results indicate that the product had no acute cytotoxic effects on cells and that, in all treatment conditions, only normal cell turnover occurred . After 4 days, cilia beating frequency at the apical surface was measured .The release of LDH to the basolateral medium, a sign of cell lysis, was assayed after 2 and 4 days of exposure to the products B. At botmeasured C. Untrea50 compared with saline control was observed, which corresponds to about a 99% lower viral titer . The application of the product was repeated daily for 4 days A,B. In cal titer A. AM-301 was shown to be effective also when inserts were infected with the SARS-CoV-2 Delta variant, with a six-fold reduction in viral titer compared with saline and matrix-treated groups on Day 3, and of six- and three-folds on Day 4, respectively B.Viral titer for the inserts not exposed to SARS-CoV-2 was below detection, indicating that the test substances and culture medium were free of viral contamination .Because viral replication was unhindered in the matrix-treated samples, we can infer that bentonite within the AM-301 formulation is primarily responsible for the effect.p < 0.001). AM-301 was able to significantly decelerate viral titer growth also in case of infection by the Delta variant, compared with inserts receiving saline solution or the matrix (p < 0.001).The data from the above prophylaxis experiments  were statistically analyzed using a linear mixed-effect model C,D. The To test AM-301’s ability to mitigate an existing SARS-CoV-2 infection of the nasal epithelium, MucilAir inserts were infected and then treated with test substances starting 24 h after infection A,B. A hiAM-301 was effective at reducing the viral titer also in the case of infection by the SARS-CoV-2 Delta variant B, reachiThe viral titer for the inserts not exposed to SARS-CoV-2 was below detection, indicating that the test substances and culture medium were free of viral contamination (data not shown).p < 0.05; Delta variant, t-value 4.08, p < 0.001) compared with inserts that received saline solution or matrix. Importantly, all experimental conditions were statistically equivalent at baseline. All data were considered valid, and no outliers were excluded.As in the prophylaxis experiment , data from the mitigation arm of the study  were also analyzed with a linear mixed-effect model C,D, whicAltogether, despite noticeable intragroup variability, these results suggest that AM-301 can mitigate an established infection even when applied several hours post-exposure to the virus, both in the cases of the SARS-CoV-2 WT and the Delta variant.To study the ability of AM-301 to prevent or reduce SARS-CoV-2 infection in the nasal mucosa, we used a well-established model of primary human nasal epithelium also for viral infections, including SARS-CoV-2: MucilAir ,30,31,32In the first part of the study, we performed in vitro safety assays  to assess potential toxicity issues of AM-301. The results displayed in In the second part of the study, we focused on the evaluation of AM-301 as a potential prophylaxis or treatment against SARS-CoV-2 infection. To investigate that, we used the same in vitro model of human nasal airway epithelium (MucilAir), receiving AM-301 application either pre-viral load B,D.The results shown in The effects exerted by AM-301 can be attributed to a mechanical, not biological, action. Indeed, AM-301 was developed to mechanically prevent the virus from contacting the nasal mucosa and infecting the upper respiratory airways and to trap or bind it for clearance by mucociliary clearance, ultimately helping to prevent a dramatic viral replication and spread in the airways. This proposed mechanism is based on the hypothesis that viral particles are bound by the complex gel mesh of AM-301 stabilized by bentonite particles and not exclusively due to the presence of polyanionic substances ,37,38. TAM-301 is a substance-based medical device that contains only excipients and inert ingredients generally recognized as safe. Clays such as bentonite, a key component of the formulation, can also be used to detoxify water of fluoride or heavy metals , and benBentonite in synergy with other formulation components confers thixotropic properties to AM-301, permitting its easy application with a nasal spray pump, which results in a protective film once it contacts the nasal epithelium. The lack of preservatives, decongestants, and other pharmacologically active molecules was intended to ensure maximal safety and, potentially, compatibility with the nasal microbiome. Further investigations to confirm this would be of particular interest. Indeed, this feature may be particularly important, since a reduction or imbalance, most recently observed also for SARS-CoV-2, in nasal microbiota diversity provoked by influenza seem to be associated with pathological conditions of the respiratory tract, such as chronic rhinosinusitis, asthma, bronchiolitis, allergic rhinitis, and otitis media ,47. TherConducting this study was challenged by several factors that may account for the noticeable intragroup variability: differences in the mucus quantities produced by the MucilAir inserts and technical difficulties in the washing procedures to evenly remove the mucus from all inserts. However, these in vitro results highlight the potential and relevance of AM-301, since the MucilAir tissue model can be considered a worst-case scenario in terms of protection from a viral infection compared with the in vivo situation. Indeed, MucilAir lacks a supportive immune system to protect against infection, and mucociliary clearance of viral particles does not occur. AM-301 was applied once every 24 h, whereas in patients, 2–3 administrations per day would be likely. Indeed, clinical data from an allergen exposure chamber study showed that AM-301 well tolerated and provided a protective effect lasting more than 3 h . The encIntriguingly, since nasal sprays such as AM-301 are not absorbed by the nasal mucosa and are discharged from the nose to the pharynx (manuscript in preparation), AM-301 may also exert its beneficial virus blocking effects in the throat. Since the oral cavity is the main production site of aerosols and airborne droplets ,50, thisCurrently, the entire world must deal with the health, social and economic damage that the COVID-19 pandemic caused. Several therapies and vaccines have been developed, allowing us to gradually return to normal life. The ability of SARS-CoV-2 to mutate quickly and the high cost of medications, vaccines, and protective measures as well as supply chain challenges make this process even more difficult in developing countries ,53. It iThe SARS-CoV-2 strain 2019-nCOV/Italy-INMI1 was obtained from the National Institute for Infectious Diseases Lazzaro Spallanzani IRCCS  . The SAR2 atmosphere) on Costar Transwell porous inserts  with MucilAir serum-free culture medium . Approximately one month after seeding, when the cultures were fully differentiated and pseudo-stratified, with basal cells, ciliated cells, and mucus cells, they were exposed to air on the apical surface and were considered suitable for experimental use. Prior to testing, MucilAir Pool cultures (hereafter “MucilAir inserts”) were cultured with 0.7 mL culture medium in the basolateral chamber and air at the apical surface; the medium was changed every 3 days. Each insert had approximately 500,000 cells.MucilAir Pool tissue cultures were obtained from Epithelix  ,55,56. MThe nasal spray formulation tested (AM-301) in this study is a medical device containing bentonite  in a matrix composed of mono-, di- and triglycerides; propylene glycol; xanthan gum; mannitol; disodium EDTA; citric; acid and water. All components are listed in the Inactive Ingredient Database of the US Food and Drug Administration (FDA), are classified as “generally recognized as safe”, or are approved for use as food additives by the FDA. The formulation is a white to light beige, aqueous gel emulsion with a pH of 6.0. AM-301 is odorless and tasteless. When we applied it to our own nasal mucosa or palmar skin, it had a soothing non-irritant, lotion-like consistency.2 [2, 10 μL of a 1:5 aqueous dilution of AM-301 or its matrix (diluted in water immediately before use) was tested per insert.Before testing, AM-301 and its matrix were brought to room temperature and vigorously agitated for 10 s. The amount of product tested on MucilAir inserts was calculated to roughly correspond to the amount that would be delivered to the nostril by a standard, commercial spray applicator (140 µL per actuation). Considering that the total adult nasal cavity surface area is 160 cm2  and that2 , 5.3 μL AM-301 was tested for potential cytotoxic effects in a series of assays to determine the viability and function of the epithelial tissues used routinely for assessing the quality of MucilAir preparations. These assays, performed by Epithelix, included an assay for transepithelial electric resistance (TEER), which measures tight junction integrity ; an assaBriefly, 3 days before use, the inserts were washed apically with 200 μL culture medium (10 min), and TEER was measured to verify their integrity. The apical medium was discarded, and the inserts were returned to the incubator for 3 days. On the first day of product testing (Day 0), the inserts were transferred to new 24-well plate with 500 μL/well fresh medium. The apical surface was treated in triplicate with 10 μL of a 1:5 aqueous dilution of AM-301 or its matrix or left untreated. The inserts were returned to the incubator for 24 h.On Day 1, 200 μL of the medium was applied apically for TEER measurements. The apical medium was removed, and the inserts were transferred to a new 24-well plate with 500 μL/well fresh medium. The inserts were treated as on Day 0 and returned to the incubator. On Day 2, 200 μL of the medium was applied apically for TEER measurements. The apical medium was removed, and the basolateral medium was collected for LDH assays. The inserts were transferred to a new 24-well plate with 500 μL/well fresh medium, treated as on Day 0, and returned to the incubator. On Day 3, 200 μL of the medium was applied apically for TEER measurements. The apical medium was removed, and the inserts were transferred to new 24-well plate with 500 μL/well fresh medium; the inserts were treated as on Day 0 and returned to the incubator. On Day 4, cilia beating on the apical surface was observed and its frequency was quantified. Then, 200 μL medium was applied apically for TEER measurements. Finally, the basolateral medium was collected and assayed for LDH. 2) using the following formula: 2 is the surface area of the insert. The assay was performed in triplicate. Normal values for MucilAir are in the range 200–600 Ω cm2 [Transepithelial electric resistance (TEER) was measured with an EVOMX epithelial volt-ohm meter (World Precision Instruments). Briefly, 200 μL of the MucilAir culture medium (34 °C) was applied to the apical surface of each MucilAir insert. The electrodes were washed with 70% ethanol and then with medium prior to insertion into the apical and basolateral media. Resistance values (Ω) were measured at room temperature; they were corrected and converted to TEER (Ω cm00 Ω cm2 .n = 3) were treated apically with 100 μL of a lysis solution for 24 h in a tissue culture incubator, and the basolateral medium was collected. For the assay, 100 μL of the medium from each test sample and from the high controls was transferred to a 96-well plate; culture medium alone was used as the low control (n = 3). The reaction solution was added (100 μL/well), and the plate was incubated for 15 min in the dark at room temperature. Then, a stop solution was added (50 μL/well), and absorbance was read at 490 nm on a plate reader. Cytotoxicity was expressed as a normalized percentage relative to the high and low controls. Normal values for MucilAir inserts are ≤5%, which corresponds to a physiological turnover of cells in culture [Lactate dehydrogenase activity in the basolateral medium was assayed using the Cytotoxicity Detection Kit . For the high control, MucilAir inserts  with a 10× objective. A Mako G-030B machine vision camera  mounted on the microscope was used to capture 256 movies (125 frames per second). The registrations were analyzed with Cilia-X software (Epithelix), which calculated the cilia beating frequency. Normal values of frequency for MucilAir are in the range of 4–8 Hz.AM-301 was subjected to a series of in vitro efficacy assays using MucilAir inserts at Texcell. The ability to prevent SARS-CoV-2 infection was tested in a prophylaxis assay, while the ability to reduce SARS-CoV-2 replication in already-infected tissues was tested after post-exposure treatment in a mitigation assay. For both assays, inserts were treated apically with 100 μL of a suspension of SARS-CoV-2 in a culture medium at a multiplicity of infection (MOI) of 0.5 for the WT strain and 0.1 for the Delta variant.In the pre-viral load assay (prophylaxis), MucilAir inserts were apically treated with test items for 10 min before exposure to SARS-CoV-2 , and viral replication was measured over 4 days with daily apical treatments with the saline solution, the matrix, or AM-301. In the experiment evaluating the ability of AM-301 to prevent SARS-CoV-2 WT infection A,C, TCID2 atmosphere; then, the apical medium was removed. The apical surface was then treated with 10 μL of a 1:5 aqueous dilution of AM-301 or of its matrix, or with 10 μL saline diluted 1:10 in water. After 10 min, without washing, the apical side was treated with 100 μL SARS-CoV-2 suspension . Infection was allowed to proceed for 3 h in a 34 °C incubator and stopped by gentle washing of the apical surface with 200 μL medium (3 times). Viral replication was assessed immediately and daily over 4 days as follows:(1)Viral sampling: 300 μL of the medium was applied to the apical surface. After 20 min at 34 °C, the conditioned apical medium was collected and stored at −70 °C until analysis.(2)Medium change: Inserts were transferred to a new 24-well plate with 500 μL/well fresh medium. The conditioned basolateral medium was collected and stored at −70 °C for possible future analyses.(3)Repeat treatment: The apical surface was treated with a product or left untreated, as described above, and the inserts were returned to the 34 °C incubator.Briefly, on Day 0, the inserts were transferred to a new 24-well plate with 500 μL/well fresh medium and washed apically by incubation with 200 μL medium for 20 min at 34 °C in a humidified 5% COTreatments were repeated daily, and sampling was performed only on Day 4.In the post-viral load assay (mitigation), MucilAir inserts were apically infected with the SARS-CoV-2 WT or Delta variant, and viral replication was measured over 4 days with daily apical treatments of physiological saline, AM-301, or its matrix In the experiment evaluating the ability of AM-301 to mitigate SARS-CoV-2 WT infection A,C, TCID2 atmosphere; then, the apical medium was discarded. The apical surface was infected with 100 μL of SARS-CoV-2 suspension ; viral suspension was added to 12 inserts per protocol, while 3 inserts served as negative viral controls. Infection was allowed to proceed for 3 h in a 34 °C incubator and stopped by gentle washing of the apical surface with 200 μL medium (three times). Viral replication was assessed immediately by the addition of 300 μL of the medium to the apical surface and incubation for 20 min at 34 °C; the conditioned apical medium (Day 0) was collected and stored at −70 °C until analysis. The inserts were transferred to new 24-well plates with 500 μL/well of fresh medium and returned to the incubator. Briefly, on Day 0, the inserts were transferred to new 24-well plates with 500 μL/well fresh medium and washed apically by incubation with 200 μL medium for 20 min at 34 °C in a humidified 5% COOn Day 1, viral replication was assessed as on Day 0 for all inserts. All inserts were transferred to new 24-well plates with 500 μL/well of fresh medium. Inserts belonging to the mitigation arm of the study were treated with AM-301, its matrix, or physiological saline and returned to the incubator.On Days 2 and 3, viral replication was assessed in all inserts; the conditioned medium (Days 2 and 3) was collected and stored at −70 °C. Inserts were transferred to new 24-well plates with fresh medium; treated with AM-301, its matrix, or saline; and returned to the incubator. On Day 4, viral replication was assessed in all inserts.5 cells/mL in DMEM plus 4% FBS) was added per well. Plates were incubated at 37 °C in a 5% CO2 humidified atmosphere for 6 days. Then, a 0.2% crystal violet solution was added to stain DNA in live, adherent cells. The tissue culture infectious dose that killed 50% of cells (TCID50) was calculated using the Spearman–Kärber method.The viral titers were determined in the conditioned medium collected from the apical side of MucilAir inserts. The samples were prediluted 1:32 in DMEM containing 0.5 mg/mL gentamicin (to avoid any possible interference of the test products on cell growth). Then, using one 96-well plate per sample, serial three-fold dilutions were made in the same culture medium with eight replicates per dilution (10 dilutions per sample); eight wells received fresh medium (negative controls). A fixed volume (50 μL) from each well was transferred to sample titration plate, and 50 μL VERO cell suspension (10t-test. The results from the TEER analyses were compared using two-way repeated measure ANOVA followed by post hoc Tukey’s test. The level of significance was set at p < 0.05. The results from the SARS-CoV-2 prophylaxis and mitigation experiments were analyzed using linear mixed-effect models with log-transformed data [Data from the LDH and CBF analyses were compared using the unpaired med data . The minmed data  as implemed data .


Punica granatum L.), a nutritious and visually appealing fruit with a diverse bioactive profile, was examined for its potential anti-apoptotic, antioxidant, and anti-inflammatory effects. A total of 40 adult male Wistar rats were allocated into four groups of 10 rats each: Group 1 was a negative-control group (CNT) and received normal saline; Group 2 was a positive-control acrylamide group and received acrylamide orally at a dose of 20 mg/kg/bw; in Group 3, the rats were supplemented with pomegranate-peel extract  orally on a daily basis for 3 weeks, administered simultaneously with the acrylamide treatment described for Group 2; Group 4 was a protective group, and the animals received the pomegranate-peel extract and acrylamide as stated for Groups 2 and 3, with the pomegranate-peel extract (P.P. extract) administered 1 week earlier than the acrylamide. The results indicate that acrylamide exposure increased the serum levels of AST, ALT, creatinine, interleukin-1 beta, and interleukin-6 in an extraordinary manner. In addition, it increased the lipid peroxidation marker malondialdehyde (MDA) and simultaneously weakened antioxidant biomarker activities  and reduced the levels of interleukin-10. The pomegranate-peel extract was shown to reduce the inflammatory blood markers of interleukin-1 beta and IL-6. Glutathione peroxidase, superoxide dismutase, catalase, and interleukin-10 were all significantly elevated in comparison to the acrylamide-treatment group as a result of the significant reduction in MDA levels induced by the P.P extract. In addition, the pomegranate-peel extract normalized the cyclooxygenase-2 (COX2), transforming growth factor-beta 1 (TGF-β1), and caspase-3 levels, with a significant upregulation of the mRNA expression of heme oxygenase-1 (HO-1), nuclear factor erythroid 2 (Nrf2), and Bcl-2. Therefore, these data reveal that pomegranate peel has anti-inflammatory, antiapoptotic, free-radical-scavenging, and powerful antioxidant activity that protects against acrylamide toxicity.The Acrylamide is a toxic compound generated under oxidative stress arising from intracellular ROS production and induced toxicity. It is frequently used in industry and generated through the heating of tobacco and foods high in carbohydrates. The exact mechanism of its toxicity is still unclear. In this study, an extract of the peels of pomegranate ( Acrylamide is a transparent, colourless, odourless, crystallized monomer . GlucoseAcrylamide can be absorbed through the digestive system, the respiratory system, or the skin . Due to Oxidative stress is the cause of acrylamide’s harmfulness. The metabolization of acrylamide by glutathione or cytochrome P450 to glicidiamine is a greater danger than acrylamide itself . The forPunica granatum L.) [One of the top seven fruits for human health is the pomegranate (atum L.) . Thyroidatum L.) . It is watum L.) . Simple atum L.) . Pomegraatum L.) ,15.Pomegranate juice has a wide range of health benefits, including antioxidant activity ,25,26,27Punica granatum Lythraceae) are two of the most important natural fruit trees in Taif. Taify pomegranate is the most widely grown pomegranate type in Saudi Arabia. It has a significant cultural, nutritional, and commercial importance [Taif is a high-altitude location with elevations ranging from 1200 to 2300 m above sea level, making it ideal for a diverse range of indigenous plant species . Grape aportance . As a reportance ,39.Given this background, we performed an experiment to investigate the hepatotoxicity of acrylamide, and to assess the protective effect of pomegranate extract and its intracellular pathway against the toxicity of acrylamide.Our experiment involved 40 male Wistar rats which were 10 weeks old. The rats had free access to food and water and were housed at room temperature. Over the course of seven days, the rats were handled manually in the Turabah University labs to adapt them to the experimental conditions. Four groups were established, with 10 rats per group. Group 1 was a negative-control group (CNT), which received normal saline; Group 2 was a positive-control acrylamide group, which was orally administered acrylamide at a dose of 20 mg/kg/bw  for 2 weOn Day 21, all of the rats were anesthetized using isoflurane and decapitated. Blood samples and liver tissues were harvested under aseptic conditions. The serum was extracted, used for chemical analysis, and stored at −20 °C. Hepatic tissue samples for RNA analysis and real-time PCR were preserved in QIAzol reagent. Bouin solution was used to preserve liver samples for histology and immunohistochemistry.In October 2020, fresh pomegranate fruits were collected from a farm in the Al-Hada region of Saudi Arabia’s Taif region. The peels were air-dried before being ground into a fine powder. The fine powder was extracted for 3 days at room temperature with 1000 mL of absolute ethanol. The extract was centrifuged at 7000 rpm for 15 min and filtered 3 times with Whatman filter paper No.1 to obtain a pure supernatant. The supernatant was evaporated at 30 °C using a Buchner funnel in a rotary vacuum evaporator. The pellet was then dissolved in a 1% aqueous solution of dimethyl sulfoxide (DMSO). The extract was kept at 4 °C until the HPLC analysis and experiments were used.® 5 µm EVO C18 100 mm × 4.6 mm  column was used, operated at 30 °C. The separation was conducted using a ternary linear elution gradient with (A) HPLC-grade water and 0.2% H3PO4 (v/v), (B) methanol, and (C) acetonitrile. Then, 20 µL of the sample was injected, and a variable wavelength detector set at 284 nm was used to recognize phenols and flavonoids. HPLC is selected to convert each component’s area under the curve compared to the area under the standards curve to a concentration (mg/L). In the case of making diluted samples, the concentration is multiplied by the dilution factor. The work was conducted at Cairo University’s Faculty of Agriculture’s Food Safety and Quality Control Laboratory (Reference Number: FSQC0007-21). See The extract was analysed, and phenol and flavonoid compounds were detected according to the protocol , where aThe concentration of phenols and flavonoids are expressed on mg/L. mAu is milli-absorbance unite.The serum levels of ALT, AST, and urea were measured using a colorimetric spectrophotometer as described in the instruction manual. The procedures described in  were useThe ELISA kits for rat IL-1β and IL-6  were used with an ELISA spectrophotometer as described in the instruction manual for each kit. IL-10 was measured using a commercial kit available from Abcam co. USA (Rat IL-10 ELISA Kit (ab100765)). The levels were calculated based on data obtained from the ELISA reader according to the instructions provided for each parameter.Total RNA was extracted from the liver samples and quantified using a spectrophotometer at 260 nm according to the manufacturer’s instructions . To make cDNA, two grams of isolated RNA was used . To quantify the expression of different hepatic genes, synthesised cDNA was amplified with SYBR Green master mix . Hepatic tissue samples were fixed in buffered formalin with a neutral pH (10%). Liver samples were processed and stained with haematoxylin and eosin .The immunohistochemical study was conducted following the method of . Tissue p-values less than 0.05 were considered to indicate statistical significance.Data are represented as the means ± SEs. The results were evaluated using one-way analysis of variance (ANOVA). For the comparison of significance between the groups, Duncan’s test was utilized as a post hoc test, and p < 0.05) in urea levels, accompanied by decreased total protein levels, in acrylamide-treated rats in relation to the other treatment groups. These effects were overcome in the rats that were simultaneously treated with pomegranate-peel extract (Group 3). In addition, p < 0.05) in the AST and ALT levels in acrylamide-treated rats in relation to the other treatment groups. The rats co-treated with P.P. extract and acrylamide showed a significant normalization of both AST and ALT levels.Our results indicated that there were noteworthy increases (p < 0.05) increases in the MDA level in acrylamide-treated rats in relation to the other treatment groups. The rats co-treated with P.P. extract and acrylamide showed a significant normalization of catalase, SOD, and GSH levels, with significant decreases in the MDA levels in relation to the acrylamide-treated rats.p < 0.05) in the levels of IL-1β and IL-6 in acrylamide-treated rats in comparison to the other treatment groups. IL-1β and IL-6 levels were significantly reduced in the rats co-treated with P.P. extract and acrylamide in comparison to the acrylamide-treated rats. Our data concerning IL-10 reveal that there was a significant decrease in IL-10 levels in the acrylamide-treated rats, which showed a significant enhancement in the rats co-treated with P.P. extract and acrylamide.Sections from normal rats displayed normal features of hepatic parenchyma organized in hepatic cords radiating from the central veins. Sections from acrylamide-treated rats c showed Reactive oxygen species (ROS) are a by-product of the natural metabolism of oxygen in mammalian bodies, and are neutralized by the antioxidant system . IncreasIn both healthy and diseased states, the liver is responsible for the organized metabolism of a variety of nutrients, including carbohydrates and toxins . The purAs confirmed by the reduced histopathological damage, the pomegranate-peel extract meaningfully restored the liver markers. This is consistent with the results in , where tAcrylamides have been found to disrupt the oxidant/antioxidant balance, leading to an increase in ROS and, ultimately, oxidative injury . In the In our study, pomegranate peel extract administration, alone or in combination with acrylamides, significantly increased the levels of SOD, GSH, and CAT, and decreased the levels of MDA in the blood. These findings agree with those in ,67, wherPunica granatum L.) is high in natural antioxidants , which have promising antioxidant properties [Pomegranate  instead of single pure polyphenols . PomegraIn DPPH radical scavenging, superoxide radical scavenging, and lipid peroxidation inhibitory experiments, carpellary membrane extract from pomegranate fruit showed a vigorous antioxidant activity . EllagicThe cellular metabolism of cytokines relies on the liver’s ability to synthesize and eliminate them . ChronicSeveral studies have shown that pro-inflammatory cytokines such as TNF-α, IFN-β, and IL-6 are increased by free-radical-mediated apoptotic mechanisms. It seems to be reasonable to hypothesise that antioxidants and free-radical scavengers could counteract this effect, which supports our findings indicating that co-treatment with pomegranate-peel extract and acrylamide overcame the effects of acrylamide concerning the release of cytokines. In addition, in a study by Toklu et al., treatment with pomegranate-peel extract reduced the levels of TNF-α and IL-1β in BDL rats, indicating that pomegranate peel reduces the oxidative liver damage caused by BDL via its antioxidant effects . AdditioOur data indicate that there was a significant decrease in the IL-10 levels in the acrylamide-treated rats, while significantly higher levels were observed in the rats co-treated with P.P. extract and acrylamide; these results agree with those of , where t4-induced liver fibrosis may be alleviated by actuating Nrf2, which is consistent with our findings [The inflammatory process is modulated by NF-κB, resulting in increases in inflammatory cytokines such as IL-1β, IL-6, and COX2 . Our resfindings . Finallyfindings , who repThis is the first study to describe, in detail, the hepatic toxicity of acrylamides in conjunction with a description of the possible ameliorative effects of pomegranate-peel extract. Our results indicate that pomegranate effectively reduced the toxicity of acrylamides in the treated rats. A possible mechanism for niaction may be related to the improvement of liver function; reduction of oxidative stress and inflammatory responses; and modulation of COX2, TGF-B1, and caspase-3, along with a significant upregulation of the expression of HO-1 in rats. Thus, our results suggest that pomegranate-peel-extract supplementation can reduce the toxicity of acrylamides.


Cocaine addiction is an acquired behavioral state developed in vulnerable individuals after cocaine exposure. It is characterized by compulsive drug-seeking and high vulnerability to relapse even after prolonged abstinence, associated with decreased neurogenesis in the hippocampus. This addictive state is hypothesized to be a form of “memory disease” in which the drug exploits the physiological neuroplasticity mechanisms that mediate regular learning and memory processes. Therefore, a major focus of the field has been to identify the cocaine-induced neuroadaptations occurring in the usurped brain’s reward circuit. The neurosteroid dehydroepiandrosterone (DHEA) affects brain cell morphology, differentiation, neurotransmission, and memory. It also reduces drug-seeking behavior in an animal model of cocaine self-administration. Here, we examined the long-lasting effects of DHEA treatment on the attenuation of cocaine-seeking behavior. We also examined its short- and long-term influence on hippocampal cells architecture (neurons and astrocytes). Using a behavioral examination, immunohistochemical staining, and diffusion tensor imaging, we found an immediate effect on tissue density and activation of astrocytes, which has a continuous beneficial effect on neurogenesis and tissue organization. This research emphasizes the requites concert between astrocytes and neurons in the rehabilitation from addiction behavior. Thus, DHEA may serve as a treatment that corrects brain damage following exposure to and abstinence from cocaine. Drug addiction is a complex disease involving mind and body, defined as the compulsion to consume a drug while losing control over the amount consumed . CocaineThe neuroadaptations following cocaine administration have been studied extensively to determine the consequences of cocaine abuse and potential addiction treatment targets. Merging reports emphasize the importance of both the glutamatergic signaling-pathway and the learning and memory circuit for switching from substance use- to substance abuse-behavior . SubstanDehydroepiandrosterone is a neuroprotective steroid that modulates neuronal function, stimulates neurogenesis in the hippocampus of rats, promotes survival of newly formed neurons, and prevents corticosterone-induced suppression . DHEA anThis study aimed to examine post-DHEA treatment’s long-term (one month) effect on drug-seeking behavior and neurogenesis in rats. We used diffusion tensor imaging (DTI), a non-invasive method, to evaluate longitudinal changes of hippocampus tissue in a month-long period of DHEA treatment during withdrawal from repeated cocaine self-administration. We examined proliferation in the short-term and then neurogenesis concurrently with the DHEA effect on drug-seeking behavior one month post-treatment.via coordinated uptake and release, hence contributing to learning and memory  were used. Rats were purchased from Envigo Inc., were maintained (in pairs) on a 12–12 h light-dark cycle (lights off at 07:00 AM) with free access to food and water. All experimental procedures were approved by the Animal Care and Use Committee of Bar Ilan University and performed in accordance with the guidelines of the National Institute of Health.Rats were trained to self-administer cocaine  for 16 days under an FR-1 schedule of reinforcement. 5 days after catheterization, rats were transferred to operant conditioning chambers  for one hour daily during their dark cycle. Each self-administration chamber (30⋅25⋅22 cm) had two levers, active and inactive, located 9 cm above the chamber’s floor. The self-administration chambers and the computer interface were built locally and controlled by a computer program written by Steve Cabilio .An active lever press generated a cocaine infusion  through the IV catheter, which was connected to an infusion pump. Throughout cocaine infusion intervals, a light located above the active lever was lit for 20 s. During the 20-s intervals, active lever presses were recorded, but no additional cocaine reinforcement was provided. Presses on the inactive lever did not activate the infusion pump and light. The number of active lever responses, infusions, and inactive lever responses was recorded. Rats were returned to their home cages at the end of the daily session.Rats subjected to DHEA (COC-DHEA group) or vehicle (COC-SAL group) injections were placed daily in the operant chamber for one hour without receiving cocaine in response to active lever pressing, until lever-pressing behavior resembled extinguished values. When the rats’ pressing on the active lever decreased by less than 80% deviation from the first day of extinction, it was assumed to be non-reinforced by cocaine. The control groups were trained to self-administer saline, subjected to DHEA (SAL-DHEA group) or vehicle (SAL-SAL group) during extinction. For the next 28 days, rats were not exposed to cocaine or operant chambers. After a total of 36 days since their last exposure to cocaine, rats received a priming injection of cocaine  before placement in the self-administration chambers (operant chamber). Active lever responses, reinforcements, and inactive lever responses were recorded.Dehydroepiandrosterone  was dissolved in 0.3 ml of dimethylsulfoxide (DMSO) and then diluted to 1 mg/ml of saline . Rats were injected i.p. with DHEA (2 mg/kg) or vehicle  90 min prior to exposure to the operant chamber. Based on previous research showing that cocaine addicts that had a twofold increase in plasma DHEA-S levels compared to other addicts were less prone to relapse and based on prior evaluation of the effect of DHEA injections on DHEA-S plasma levels in rats, we found that the optimal DHEA dose to achieve a twofold increase in DHEA-S plasma levels was 2 mg/kg in cocaine self-administrating rats .Rats were housed under standard conditions and injected i.p. with BrdU  3 injections at 4-h intervals for one day after reaching stable maintenance levels. They continued to live in this environment for 24 h or 28 days after the last injection. Then, they were euthanized and perfused transcranial, first with PBS and then with 4% paraformaldehyde.Phosphate buffer (PBS) perfusion was performed to the left ventricle in the rat heart until the blood washed out through the right atrium, then 4% paraformaldehyde was injected in the same manner for brain fixation. Brains were removed, post-fixed overnight, and equilibrated in phosphate-buffered 30% sucrose. Free-floating, 40-μm-thick coronal hippocampal sections were collected on a freezing cryostat and stored at 4°C before immunohistochemistry.For BrdU staining of rat specimens, tissue sections were washed with PBS, incubated in 2N HCl at 37°C for 30 min, and then blocked for one h with blocking solution . The tissue sections were stained overnight with specified combinations of the following primary antibodies: rat anti-BrdU  and mouse anti-NeuN . Secondary antibodies used for both rat tissues were Cy-3-conjugated donkey anti-rat  and Cy-2 donkey anti-mouse .Astrocytes were immunohistochemically stained for S100β. Tissue sections were rinsed in PBSx1 + 0.1% triton and then blocked in 20% normal horse serum + 0.1% triton + PBSx1. s100β antibody  with PBSx1 + 2% normal horse serum + 0.1% triton solution. The slices were stored at 4°C overnight. The second (fluorescent) 488 alexa antibody (green) was introduced at a ratio of 1: 1000  in PBSx1 + 0.1% triton solution for one hour. Slices were washed again and stained with fluorescent nucleus by DAPI  in PBSx1 + 0.1% triton solution for 10 min.For microscopic analysis, we used a fluorescent microscope (40× magnifications). Neurogenesis in the dentate gyrus was evaluated by counting the cells that were double-labeled with BrdU and NeuN. We counted the number of labeled cells in nine coronal sections (360 μm apart) per rat brain, stained them, and mounted them on coded slides. To obtain an estimate of the total number of labeled cells per dentate gyrus, we multiplied the total number of cells counted in the selected coronal sections from each brain by the volume index .For astrocyte analysis, we used a Leica SP8 confocal microscope; excitation wavelengths: 488 nm, 405 nm, using a white-light laser. A 3D image of the ventral and dorsal dentate gyrus was performed; Scale bar is 50 μm. All raw images were analyzed using Leica’s analysis software.The rats were implanted with intravenous Silastic catheters  into the right jugular vein . The catDiffusion-tensor imaging (DTI) is a technique in which diffusion is measured in a series of different spatial directions. The shape and orientation of the diffusion tensor are determined at each pixel.b-value shell at 1000 s/mm2; and one image with a b-value of 0 s/mm2 (referred to as b0). Geometrical parameters were: 20 slices, each 0.48 mm thick (brain volume) and with in-plane resolution of 0.2 × 0.2 mm2 . The imaging protocol was repeated three times for signal averaging and compensated for acquisition in which significant head motion was observed. Each DTI acquisition lasted 4.5 min, and the entire MRI protocol lasted about 20 min.MRI was performed at Tel-Aviv University using a 7T MRI scanner  with a 30-cm bore and a gradient strength of up to 400 mT/m. The MRI protocol included diffusion tensor imaging (DTI) acquisition with a diffusion-weighted (DW) spin-echo echo-planar-imaging (EPI) pulse sequence with the following parameters: TR/TE = 4000/25 ms, Δ/δ = 10/4.5 ms, four EPI segments, and 15 non-collinear gradient directions with a single Image analysis included DTI analysis of the DW-EPI images to produce the fractional anisotropy (FA), apparent diffusion coefficient (ADC), and radial and axial diffusivity indexed maps (RD). DTI enables quantifying the motional anisotropy of water molecules and can provide images with high contrast toward the white matter. Lower FA values may indicate lower tissue directionality and organization or reduced axonal and myelin density. Higher ADC values may indicate reduced tissue density and integrity.b0 images initially underwent bias correction. (b) The b0 image for each rat was co-registered with the b0 template (using a 6-parameter rigid-body transformation). The co-registration parameters were then applied to the different DTI indexed maps . (c) The registered FA maps were normalized to the FA template  using a 12-parameter affine non-linear transformation and 0.2 mm smoothing (which was used only for the parameter estimation procedure and not smoothing of the output image). The normalization parameters were then applied on all DTI indexed maps, including the FA, ADC, and radial and axial diffusivities. (d) The normalized indexed maps were smoothed with a 0.3-mm Gaussian kernel.The DTI analysis was implemented in Matlab  using in-house software . For stat-test was used as appropriate. Data are presented as mean ± SEM. Results were considered significantly different if P < 0.05.For the behavioral data , we applied one-, two-, or three-way ANOVA with or without repeated measures, followed by Bonferroni’s multiple comparisons test. The histological data  were analyzed by one-way ANOVA, followed by Bonferroni’s multiple comparisons test. DTI data were analyzed by one-way ANOVA with or without repeated measures, followed by Bonferroni’s multiple comparisons test, or a two-tailed Student’s unpaired To examine long-term effects of DHEA treatment, we trained rats daily for one hour of cocaine self-administration until reaching constant drug consumption levels . After rPrevious research in our lab found that high doses of cocaine (1.5 mg/kg) self-administration reduce proliferation (newly formed cells) and neurogenesis (newly formed cells that mature to neurons) . Since DSignificantly more newly formed cells . This agrees with Becker’s theory, according to which newly formed neurons support adaption to changed environmental circumstances, and as such, the flexibility of new neurons to merge into new properties is raised by the microenvironment , Bar-Ilan University Ramat-Gan 52900.HA-L contributed to behavioral, histological experiments and data analyses, and wrote the manuscript. OC and ES participated in the former design of the experiments and critically assisted with different aspects of the behavioral experiments. TB and HP-N assisted with the behavioral experiments. IG contributed to the surgical procedures and animal care and rehabilitation. AJ assisted with the microscopy data analyses. RM and AW provided critical interpretations revision of the manuscript. GY contributed to the research concept and design, interpretation of the results, and critical revision of the manuscript. All authors reviewed the content and approved the final version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background and Purpose: An endovascular recanalization is an alternative option for symptomatic intracranial atherosclerotic steno-occlusive disease (ICAD). Accurate non-invasive alternatives to digital subtraction angiography (DSA) for follow-up imaging after endovascular treatment are desirable. We aimed to evaluate the image quality and diagnostic performance of high-resolution magnetic imaging in follow-up using DSA as a reference.Materials and Methods: From January 2017 to June 2021, data from 35 patients with 40 intracranial steno-occlusive lesions who underwent endovascular recanalization and received high-resolution magnetic resonance (HR-MR) follow-up were retrospectively collected in our prospective database. Studies were evaluated for the quality of visualization of the vessel lumen, restenosis rate, and accuracy of high-resolution magnetic resonance (HR-MR) with DSA used as the reference standard. Intraclass correlation coefficient (ICC) analyses were performed to assess the agreement between the two different readers.Results: In total, 40 intracranial steno-occlusive lesions in 35 patients, with 34 lesions undergoing balloon angioplasty [including 16 drug-coated balloons (DCBs)] and 8 lesions undergoing stenting were enrolled. The median age was 63.6 years (IQR 58.5–70.0 years), and the mean imaging follow-up time was 9.5 months (IQR 4.8–12.5 months). The median degrees of preprocedural and residual stenosis were 85.0% (IQR 75.0–99.0%) and 32.8% (IQR 15.0–50.0%), respectively. Intracranial periprocedural complications occurred in 1 (3.6%) patient. In the case of a stainless-steel stent (n = 1), there was a signal drop at the level of the vessel, which did not allow evaluation of the vessel lumen. However, this was visible in the case of nitinol stents (n = 7) and angioplasty (n = 34). The overall restenosis rate was 25.8% (n = 9). The DCB subgroup showed a lower rate of restenosis than the percutaneous transluminal angioplasty (PTA) subgroup [5.3% (2/13) vs. 35.7% (5/14)].Conclusion: High-resolution magnetic resonance may be a reliable non-invasive method for demonstrating the vessel lumen and diagnostic follow-up after endovascular recanalization for ICAD. Compared with MR angiography (MRA), HR-MR showed a higher inter-reader agreement and could provide more information after endovascular recanalization, such as enhancement of the vessel wall. Intracranial artery disease (ICAD) is one of the major causes of ischemic stroke and neurologic symptoms, especially in Asians –4. EndovLumenography, such as digital subtraction angiography (DSA), CT angiography (CTA), and MR angiography (MRA), has been essential and widely used in the diagnosis of ICAD and follow-up imaging . Among tHigh-resolution magnetic resonance (HR-MR) imaging has been recently introduced as a minimally invasive-advanced imaging technique for directly identifying the intracranial arterial wall , 12, whiThis retrospective study was approved by our institutional review board and information of the patient was anonymized and reidentified before the assessment.The studies involving human participants were reviewed and approved by the institutional ethics committee at Beijing Hospital. The patients provided written informed consent to participate in this study. Prior to the intervention, patients gave their informed consent to the operation. Patients (≥18 years) with symptomatic, intracranial high-grade stenosis (WASID ≥ 70%) or occlusion and elective [ipsilateral hypoperfusion ] endovascular recanalization between January 2017 and June 2020 who underwent HR-MR follow-up were included in this retrospective data analysis. Patients with hyperacute (0–24 h) stroke or with asymptomatic steno-occlusive lesions and without follow-up were excluded.Demographical, clinical, angiographical, and periprocedural data were collected. The primary follow-up outcomes were angiographic restenosis, ISR, and recurrent ischemic events. Restenosis or ISR was defined as >50% stenosis within or immediately adjacent (within 5 mm) to the treated segment and >20% absolute luminal loss. Symptomatic restenosis was defined as restenosis associated with ischemic symptoms of the offending vessel territory.Digital subtraction angiography was performed for all the patients and the strategy of endovascular treatment was decided according to the site and characteristics of the target lesions and based on the experience and preference of the operators. In general, drug-coated balloons (DCBs)  with balloon predilatation were selected in lesions with tortuous arterial access or with a significant mismatch in the diameter between the proximal and distal segments or lesions in vessels with small reference diameters. Balloon dilation (BD) alone  was performed in patients with more tortuous arterial access for which DCB or stenting was considered improper by operators. Stenting  was preferred in lesions with straightforward arterial access or was deployed for the treatment of dissection after balloon dilation.Endovascular procedures were performed by experienced neurointerventionists with the patients under general anesthesia. Prior to the intervention, all the patients were under dual antiplatelet therapy (DATP) with aspirin and clopidogrel. An intravenous heparin regimen was administered to maintain an activated clotting time between 250 and 300 s during the procedure. A 6F-guiding catheter was introduced through the common femoral artery and guided into the ICA or VA proximal to the target lesion. The precise length and diameter of the lesion were assessed by three-dimensional (3D) DSA prior to the recanalization procedure. Under the fluoroscopic guidance, a microwire was first passed through the intracranial steno-occlusive lesion. Then, one of the three kinds of interventional procedures  was performed based on the experience of the operators. The choice of strategy is as described earlier.Dual antiplatelet therapy was maintained for at least 6 months and aspirin or clopidogrel alone was continued daily afterward. Long-term management of individual medical risk factors, such as blood pressure, cholesterol, and diabetes mellitus, was implemented.High-resolution magnetic resonance was performed with a 3.0-T MR scanner  with a 16-channel NV coil. The HR-MRI sequences included 3D time of flight MRA and pre and postcontrast T1W imaging (VISTA). The parameters were as follows for time-of-flight MRA: repetition time = 25 ms; echo time = 3.45 ms; field-of-view = 180 mm × 180 mm; and acquired resolution = 0.55 mm × 0.55 mm × 1.1 mm. For T1W imaging, the parameters were: repetition time = 800 ms; echo time = 18 ms; field of view = 200 mm × 180 mm × 40 mm; and acquired resolution = 0.6 mm × 0.6 mm × 0.6 mm. Gadoteric acid meglumine  was intravenously injected (0.1 mmol/kg of bodyweight). T1W imaging was repeated 5 min after injection.Digital subtraction angiography was performed in 4 vessels using a biplane system, including high-resolution 3D rotational angiography . Transfemoral access was used, and selective injection of a contrast medium, either iohexol  or iodixanol , was performed at a rate of 4 ml  or 3 ml  per second using an injector . The parameters were as follows: matrix, 1,024; FOV, 310 mm.Two neuroradiologists independently assessed images on the basis of the PACS workstation  for MR images and the interventional workspot  for 3D rotational DSA data.The image quality of HR-MR was first assessed by an experienced reviewer using a 3-point scale, where 1 = poor [low signal-to-noise (SNR) and obscured vessel wall or lumen boundaries], 2 = fair , and 3 = good . Images with an image quality of 1 were excluded from further analyses.The following parameters were measured: (1) the arterial luminal diameter . MR data were measured manually in 0.6-mm-thick cross-sections reformatted from HR-MR and 0.6-mm-thick cross-sections from 3D tetralogy of fallot (TOF). Data of DSA were collected from 3D reconstruction of images ; (2) stenosis degree (%) calculated according to the Warfarin–Aspirin Symptomatic Intracranial Disease trial criteria; (3) presence and change of wall enhancement at the site of steno-occlusive lesions in HR-MR. According to the enhancement in the follow-up images compared with the preprocedural images, the images were evaluated as: increase, little change, or less .p <0.05 was considered to indicate statistically significant.Continuous variables are presented as the mean ± SD and categorical variables are reported as the frequencies (percentages), as appropriate. For continuous variables, the agreement between HR-MR and DSA was assessed by using the intraclass correlation coefficient (ICC) and the Bland–Altman analyses. The level of agreement was categorized as follows: poor (ICC = 0–0.20), fair (ICC = 0.21–0.40), moderate (ICC = 0.41–0.60), good (ICC = 0.61–0.80), and excellent (ICC = 0.81–1.00). Differences between proportions were assessed by chi-squared analysis. All the statistical analyses were performed using R Statistical Software version 3.6.2 . Overall, 40 intracranial steno-occlusive lesions in 35 patients , with 32 lesions undergoing balloon angioplasty (including 16 DCB) and 8 lesions undergoing stenting, were identified. Most treated lesions were located in the anterior circulation. The median preprocedural and residual degrees of stenosis were 85.0% (IQR 75.0–99.0%) and 32.8% (IQR 15.0–50.0%), respectively. In one patient, dissection of the M2 segment of the MCA with consecutive subarachnoid hemorrhage occurred during the interventional maneuver. However, the patient recovered completely from this incident. The median radiological follow-up was 9.5 months .n = 9). Of these 9 patients with restenosis, 2 patients were in the stent group . The other 7 cases were in the angioplasty group . One patient in the stent group suffered from symptomatic restenosis and he received further revascularization with the insertion of another stent in the intracranial vertebral artery. The other 8 patients were asymptomatic and received medical treatment. The DCB subgroup showed a lower rate of restenosis than the percutaneous transluminal angioplasty (PTA) subgroup [12.5 (2/16) vs. 31.2% (5/16)] ] .The overall image quality for HR-MR was 2.8 ± 0.4 . In one case with a stainless-steel stent , there was a signal drop at the level of the vessel, which did not allow evaluation of the vessel lumen .In 39 lesions with sufficient image quality, two observers measured the diameter of the stenosis and reference vessel. The ICCs of the HR-MR group were 0.956 and 0.973, and the ICCs of the TOF–MRI group were 0.890 and 0.962, respectively. The Bland–Altman plots of the diameter of the stenosis and reference vessel between reader 1 and reader 2 are shown in Of the 40 arteries, 16 arteries were followed-up by DSA. The agreements in the quantitative measurements between HR-MR and DSA and between TOF-MR and DSA are given in Using DSA as the reference, the sensitivity; specificity; positive predictive value; negative predictive value; and accuracy of HR-MR for detecting stenosis at >50, >70%, and restenosis are presented in p = 0.06].Of the 28 stenotic lesions with both the preprocedural and follow-up HR-MR images, wall enhancement was seen in 22 (78.6%) at baseline. During follow-up, the enhancement weakened in 15 lesions and remained constant in 12, but was clear in 1 case. Compared with the PTA subgroup, more lesions weakened in the DCB subgroup [75.0 (9/12) vs. 36.4% (4/11), This study found that HR-MR had good accuracy for the assessment of intracranial reference vessels and lesion vessels in the follow-up of patients with ICAD after EVT. The measurement results of HR-MR were highly consistent with DSA. Accurate lumen information can be provided even after most stents are placed. In addition, HR-MR can also provide tube wall images, which is helpful to further evaluate the postoperative situation. Therefore, HR-MR may be a useful imaging option comparable to DSA for the evaluation of stenosis and the detection of restenosis after EVT.R >0.8) with DSA regarding the degree of stenosis and the minimal luminal diameter. Using DSA as a reference, HR-MR performed a high level of sensitivity and specificity at diagnostic of stenosis. Compared with TOF-MR, HR-MR seems to have higher interobserver reproducibility and agreement with DSA. This may be because HR-MR can provide better resolution and visualize the boundaries of the vessel wall that can help readers distinguish the vascular boundaries.In this study, we evaluated the consistency of HR-MR between the two readers. Our results showed excellent interobserver reproducibility for the measurement of the lumen diameter of stenosis and reference vessels (ICC > 0.9). At the same time, HR-MR showed good agreement (ICC > 0.9) and significant correlations  rather than just angioplasty.There are several limitations in this study. First, a retrospective study with a small number of cases may have resulted in limitations in selection bias and statistical power. The accuracy of HR-MR has yet to be confirmed by data from larger series. Second, not all follow-up images were 3D. In 2D images, the eccentricity of the stenotic lesion may have biased the lumen diameter measurement. Third, almost all the cases in this study were atherosclerotic lesions. The conclusions drawn from this series of cases are not necessarily applicable to cases of other causes, such as dissection. The imbalance of analyzed arterial segments may have led to selection bias. However, stratified analysis was not available due to the small sample size. Despite these limitations, we believe that our study reflects exploration in actual clinical practice. We hope that further research can be extended to larger samples and other diverse etiologies and apply HR-MR to the clinical prognosis field.High-resolution magnetic resonance can provide high-resolution images of both the vessel wall and lumen. It could be a reliable non-invasive method for demonstrating the vessel lumen and diagnostic follow-up after endovascular recanalization for ICAD.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Institutional Review Board of Beijing Hospital. The patients/participants provided their written informed consent to participate in this study.JW and SZ reviewed the image data. All authors contributed to the article and approved the submitted version.This study was funded by the Beijing Hospital Clinical Research 121 Project (BJ-2018-086 and BJ-2018-202), Capital's Funds for Health Improvement and Research (2020-4-4053).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Our study showed that nature-based activities during lunch breaks could enhance office workers’ mental health in general, but the effects could be short-lived, calling for regular nature-based programs in occupational settings.This study aims to test the associations of nature contact with multiple dimensions of psychological functioning. A total of 90 university employees were randomly assigned to spend their lunch breaks on (1) a 30-min structured protocol of nature contact and (2) a control group for 10 consecutive weekdays. Psychological distress, psychological well-being, and work-related psychological outcomes were assessed at baseline (T1), the day after the 10-day intervention (T2), and three months after the intervention (T3). Mixed-design analyses of variance (ANOVA) were conducted. Overall, significant intervention effects were found in the structured protocol of nature contact on psychological distress, well-being outcomes, and work engagement (all  There is growing interest in the potential benefits of nature contact for mental health and work-related outcomes in recent years. Nature in the broadest sense can be defined as areas that contain elements of living plants, animals, geological processes, and other features of the earth . ContactNature contact could be classified into three forms: outdoor, indoor, and indirect . OutdoorThe level of engagement has also been found to be directly proportional to health benefits. Higher levels of engagement in nature are consistently related to higher levels of positive affect and well-being across populations ,17. AlthOne possible way to enhance engagement is through multi-sensory stimulation. However, most research on nature’s health benefits has focused on a single sensory pathway of nature contact, especially the visual sense, with low levels of engagement . For exaHumans are multisensory, and it is the multiple contents of nature that contribute to better health ,24. HumaFinally, hands-on activities of playing and learning that involve physical touch of natural elements have been found to be associated with higher levels of confidence, motivation, and focused attention among children  and posiThe workplace has been recognized as one of the most significant social contexts for addressing mental health issues and promoting mental wellness ,35. DespThe aim of the present study is to investigate the effectiveness of a nature-based intervention on adaptive psychological functioning. Most previous studies focused on the passive reception of sensory information from the natural environment, particularly visual objects in nature. In fact, both the level of engagement and the number of sensory processes should be taken into account for assessing the health benefits of nature contact and designing nature-based mental health interventions. In particular, we administered the interventions among full-time office workers and assessed different dimensions of psychological and work-related outcomes.We compared a structured protocol with a high level of nature engagement and involvement of multiple sensory processes with a waitlist control group. We expected that participants in the structured protocol of nature contact will report higher levels of mental health and more positive work-related functioning immediately and three months after the interventions.www.clinicaltrials.gov (NCT05276245). Overall, 107 employees were recruited from The Education University of Hong Kong. Recruitment advertisements were disseminated to all administrative staff and research staff by email and posted on the university intranet. Inclusion criteria were (1) 18 years or older, (2) Cantonese speaking, and (3) full-time employment. Data were collected in two periods, November 2019 to May 2020 and February to June 2021. Eligible participants were randomly allocated to a (1) structured protocol of nature contact and a (2) control group. Four participants from the intervention group and three participants from the control group were excluded because they did not complete the required tasks. The final sample consisted of 90 eligible participants . A total of 48 participants took part in the structured protocol of nature contact  and 42 in the waitlist control condition .This study is a randomized controlled trial that was registered at Ethical approval was obtained from the Human Research Ethics Committee of The Education University of Hong Kong. The final sample of 90 eligible participants was asked to give informed consent after being fully apprised of the study aim and design. All participants were arranged to spend their lunch break on a (1) 30-min structured protocol of nature contact or a (2) control group  on 10 consecutive weekdays. Before the experiment started, all participants were told to eat their lunch in office. Then, participants who were randomly assigned to the intervention group were instructed to leave their offices and spend their lunch break on a 30-min structured protocol of nature contact, whereas those who were randomly assigned to the waitlist control group were instructed to stay at the office to spend their lunch break. In addition, the waitlist control group was told to wait for at least three months before they took part in the structured protocol of nature contact. All participants were instructed to fill out an online questionnaire before the intervention (T1), the day after the 10-day intervention (T2), and 3 months after the intervention (T3).Several procedures were taken to ensure commitment and reduce non-response among participants . First, This structured protocol was a 10-day nature-based program with a high level of nature engagement and involvement of multiple sensory processes. The program was held in lunch breaks between 12:00 noon and 2:00 p.m. Each session consisted of 30 min in the Eco Garden of the Education University of Hong Kong. Activities of this program included walking, ecological photography, sketching butterflies, planting vegetables, drinking herbal tea, observing birds, and taking a nap in nature. Each activity corresponded to specific sensory pathways. A standardized proforma was used to obtain participants’ demographic information, including age, gender , marital status , education level , monthly household income, , and medical histories of psychiatric conditions.Anxiety symptoms. The Chinese version of the 7-item Generalized Anxiety Disorder (GAD-7) was used to measure anxiety symptoms at all time points . ParticiDepressive symptoms. Depressive symptoms were measured using the Chinese version of the Patient Health Questionnaire-9 (PHQ-9)  at T1, TPerceived stress. Perceived stress was assessed at all time points using the Chinese version of the 10-item Perceived Stress Scale (PSS-10) . ParticiSubjective well-being. The WHO-Five Well-Being Index (WHO-5) consisted of five positively worded items . ParticiSatisfaction with life. Satisfaction with Life Scale (SWLS)  was usedPositive affect. The 10-item positive affect subscale of Chinese Affect Scale (CAS-PA)  assessedBurnout. Burnout was measured using the Chinese version of the 14-item Maslach Burnout Inventory-General Survey (MBI-GS)  at all tWork engagement. The Chinese version of the 17-item Utrecht Work Engagement Scale (UWES)  was usedp < 0.05 was used to determine statistical significance. To begin with, we tested whether there were differences in demographics or between dependent variables at baseline. Repeated-measures Analyses of Variance (ANOVAs) were then used to analyze the changes in each outcome across time in the two different groups. Group  was the between-subject factor, whereas timepoint  was the within-subject factor. We assessed both the main effects of group and timepoint and the group × timepoint interaction effects. Changes in dependent variables between T1 and T2 were considered as “intervention effects”, whereas changes between T1 and T3 as “post-intervention effects”. We also used one-way ANOVA to assess whether there were significant differences between the program group and the control group in each outcome at T2 and T3 separately.All analyses were conducted using IBM SPSS Statistics version 26.0 . A two-tailed significance level of p > 0.05), except for the gender ratio ; 80 (88.9%) were female. A total of 84 (93.3%) reported receiving tertiary education or above, and 6 (6.7%) received secondary education. There were no differences in the main sample characteristics between the intervention groups and the control group  = 5.832, p = 0.018) and perceived stress  = 7.213, p = 0.009), whereas there were significant main effects of timepoint on anxiety symptoms  = 3.628, p = 0.032) and perceived stress  = 7.754, p = 0.001), as well as marginally significant on depressive symptoms  = 3.019, p = 0.055) , but the effect remained relatively stable at T3 (p = 0.211) (Main effects of group was significant on depressive symptoms (= 0.055) . The gro= 0.211) .p < 0.01). In addition, the program group also demonstrated significant lower level of perceived stress than the control group at T3  = 6.825, p = 0.002), satisfaction with life  = 5.865, p = 0.041), and positive affect  = 7.332, p = 0.001). In addition, the group × timepoint interaction effects were significant for subjective well-being  = 6.067, p = 0.004) and positive affect  = 4.577, p = 0.035). Overall, subjective well-being, satisfaction with life, and positive affect were consistently higher at T2 compared to T1 (p < 0.01). Post-intervention effects  was found only on satisfaction with life, p = 0.018. Regarding the group × timepoint interaction, the program group experienced an increase in positive affect at T2 relative to T1 (p = 0.000), but there was a decline in positive affect at T3 compared to T2 (p = 0.016). The results are summarized in Repeated measures ANOVAs of well-being-related outcomes  showed that the main effects of group were non-significant, all p > 0.05 . There wp = 0.005, see Although no main effects of group on all outcomes were found across timepoints, additional one-way test showed that subjective well-being was significantly higher in the program group than the control group at T2  = 9.035, p = 0.003, whereas significant main effects of timepoint was found in burnout only, F = 4.630, p = 0.011. No significant group × timepoint interaction effects could be found across both work-related outcomes, all p > 0.05, see p = 0.015, see p = 0.011), whereas scores of cynicism were significantly increased at T2 compared to T1 in the control group (p = 0.011), see p < 0.05  = 9.035, p = 0.003, whereas the main effects of timepoint  = 2.451, p = 0.094) and the group × timepoint interaction effects  = 1.984, p = 0.145) were both non-significant. The one-way ANOVA test showed that work engagement was higher in the program group than the control group at both T2 (p = 0.001) and T3 (p = 0.017). The results are summarized in For work engagement, the main effects of group were significant, In this study, a randomized controlled trial was designed to examine the effects of contact with nature on employees’ mental health and work-related functioning. We compared (1) a structured protocol with a high level of nature engagement and involvement of multiple sensory processes (the program group) with (2) a control group across three timepoints. As hypothesized, notable intervention effects  on positive mental health and work-related functioning (except for burnout) were identified in the program group, consistent with previous evidence on the positive associations between nature contact and mental health ,5. In adOur findings were consistent with the positive link between multisensory immersive environments and greater perceived stress recovery  and satiThe major difference between our study and previous studies e.g., ,22,23) i,23 i22,2Contrary to the significant time effects on mental health outcomes in the control groups in previous similar studies ,61, outcConcerning the nonsignificant difference in burnout scores in the program group between T1 and T2, one possible explanation is that burnout could be the result of relatively longer-term experiences that cannot be easily changed within a short period of time. Burnout has been understood as a response to chronic emotional stressors at work and a slow process of progressive exhaustion . We noneTaking together the significant effects of the structured program group on reducing employees’ distress and enhancing employees’ well-being throughout the working day, it is suggested that positively experienced nature-based activities during lunch breaks may help employees cope with job stress and maintain their well-being throughout the day. Our findings also shed light on the importance of instilling nature contact in work settings, especially in urban cities such as Hong Kong. Workplace stress affects not only employees’ health and well-being but also organizational functioning . In addiSeveral limitations should be considered in evaluating the current findings. First, the intervention period in this study was short. As mentioned above, we did not find significant results for trait-like outcomes such as burnout. In addition, the intervention effects on most of the outcomes were short-lived. Future randomized control trials could be designed with longer intervention and follow-up periods. Second, the sample in our study was mainly women with high education levels. Similar intervention effects have been observed in organizational research that was predominantly conducted among highly educated women ,41. ExteNotwithstanding the limitations, this randomized controlled trial was designed with the concepts from environmental psychology and organizational psychology to investigate the effectiveness of a structured protocol of nature contact in enhancing mental health and work-related functioning. The consistent overall effect on psychological distress, well-being, and work engagement immediately after the intervention period suggests a consistent positive association between active contact with nature and mental health. To achieve more sustainable benefits of nature-based interventions, longer interventions and follow-up periods are needed. It is also suggested that landscape design, planning, and management should pay more attention to the need for contact with nature in work settings.